The opposing effects of norepinephrine and acetylcholine on the contractile force of the heart are believed to result from their respective abilities to regulate the activity of the voltage-sensitive Ca2+-channel via phosphorylation and dephosphorylation reactions. A cyclic AMP-dependent phosphorylation of either the Ca2+-channel itself, or a protein that regulates the Ca2+-channel, is believed to activate the channel, while dephosphorylation of this component is believed to inactive the channel. The identity of the phosphorylated component is not known. The ultimate aim of this proposal is to elucidate the mechanisms involved in the regulation of cardiac Ca2+-channels by phosphorylation/dephosphorylation processes. In order to achieve this goal it is necessary to be able to identify the membrane proteins that comprise and/or regulate the cardiac Ca2+-channel. We will purify the cardiac Ca2+-channel in order to identify its protein composition and determine its subunit structure. This will be made possible by using radiolabeled Ca2+-channel antagonists as specific biochemical probes of the channel. Purification of the channel will be achieved using a series of steps including affinity chromatography, ion-exchange and hydrophobic chromatography, and sucrose density gradient centrifugation. The properties of the purified protein will be ascertained in order to demonstrate that it is the Ca2+-channel. Once the components of the channel are identified, we will determine which of these components are phosphorylated and dephosphorylated in situ in 32p-loaded hearts slices. The effects of adrenergic and cholinergic agonists, as well as changes in intracellular Ca2+-, on the phosphorylation of the Ca2+-channel in situ will be determined. The nature of the phosphorylation and dephosphorylation reactions occurring in situ will be elucidated by performing in vitro studies using purified components. The effects of phosphorylation on Ca2+-channel activity will be determined in in vitro assays designed to measure 45Ca2+ flux through the reconstituted channel. In addition the effects of phosphorylation on the ability of the channel to interact with inhibitory and activator ligands will be determined. The results obtained will provide an understanding, at the molecular level, of the biochemical events involved in the regulation of the cardiac Ca2+-channel by phosphorylation and dephosphorylation mechanisms.